Effects Of Different Culture Conditions On Callus Induction And The Total Flavonoids Content Of Scutellaria Barbata D.Don

Scutellaria barbata D. Don is a perennial herb plant and the entire plant can be used as medicine. It is a famous antitumor Chinese medicine with important medicinal value, and the flavonoids is the main active substances of S. Barbata.This paper launched a series of studies about callus induction by plant culture techniques for the improvement of the total flavonoids content. Single-factor experiment was used to optimize callus induction conditions and the total flavonoids content was increased. The results of this study reveal the relationship between callus culture and different culture conditions, and lay a solid foundation for further studies to explore higher total flavonoids content.In this study, the in vitro stems of S. Barbata plants were used as the plant materials. Firstly, the different light conditions were investigated, and the optimun light condition was determined. Then the effects of auxin, cytokinin, gibberellin, spermine and sucrose on callus induction and the total flavonoids content were studied by single-factor experiment.(1) Different light conditions (16 h/8 h light four weeks, light two weeks alternates darkness, and darkness four weeks) were investigated, and different concentration of 6-BA (0.0025,0.0045 and 0.0090 mmol/L) and NAA (0.0025,0.0055 and 0.0080 mmol/L) were added in MS medium. The results showed:The highest callus induction rate (97.917%) and maximum dry weight (0.467 g) were obtained at the concentration of 0.0090 mmol/L 6-BA and 0.0055 mmol/L NAA under light treatment. At this concentration, the total flavonoids content reached 4.791%, which was 40.870% and 60.503% significantly higher than the highest value of other two groups (alternates was 3.401% and alternates was 2.985%). So light treatment was the best culture condition in this experiment.(2) The results of different combination treatment with auxin and cytokinin showed: ① The combination of 2,4-D and 6-BA:We obtained the maximum callus induction rate (79.169%) and the maximum dry weight (0.438 g) treated with 0.0025 mmol/L 2,4-D alone. And the highest total flavonoids content was 3.483%(0.0045 mmol/L 2,4-D+0.0045 mmol/L 6-BA). ② The combination of IB A and 6-BA:The best combination for induction of callus (85.417%) and dry weight (0.589 g) was 0.0045 mmol/L IBA and 0.0075 mmol/L 6-BA. The highest total flavonoids content was 4.247%(0.0025 mmol/L IBA+0.0075 mmol/L 6-BA). ③ The combination of IBA and KT:The callus was not induced of all treatments. The regeneration tissues were determined for the total flavonoids content and the highestwas 3.578%(0 mmol/L TDZ+0.0045 mmol/L KT).④ The combination of IBA and TDZ:The maximum callus induction rate (87.500%) was induced using MS medium with 0.0075 mmol/L IBA and 0.0025 mmol/L TDZ, the maximum dry weight (0.451 g) treated with 0.0050 mmol/L IBA and 0.00425 mmol/L TDZ. And the highest total flavonoids content was 5.622%(0.0025 mmol/L IBA+0.0045 mmol/L TDZ).⑤ The combination of 2,4-D and KT:The most efficent callus induction (50.000%) was achieved with stems cultivated on MS medium supplemented with 0.0045 mmol/L 2,4-D and 0.0065 mmol/L KT, the maximum dry weight (0.201 g) was obtained by 0.0070 mmol/L 2,4-D and 0.0045 mmol/L KT. And the highest total flavonoids content was 3.768%(0.0045 mmol/L 2,4-D+0.0045 mmol/L KT).⑥ The combination of 2,4-D and TDZ:The best callus induction rate (58.333%) was achieved using MS medium with 0.0045 mmol/L KT. The maximum dry weight (0.666 g) and the highest total flavonoids content(1.937%) was obtained by 0.0065 mmol/L KT alone.⑦The combination of NAA and TDZ:The highest callus induction rate (68.750%) was obtained in the medium with 0.0045 mmol/L TDZ, and the maximum dry weight (0.658 g) was obtained in the medium with 0.0025 mmol/L TDZ. The highest total flavonoids content (2.803%) was treated with 0.0065 mmol/L TDZ alone.⑧The combination of NAA and KT:In combination 0.0080 mmol/L NAA and 0.0065 mmol/L KT was the best for callus induction (23.542%),0.0025 mmol/L NAA and 0.0025 mmol/L KT was best for the dry weight (0.561 g). And the highest total flavonoids content was 2.208%(0.0025 mmol/L KT).The results showed that KT promoted the regeneration of seedlings, but the effects of KT on callus induction and the total flavonoids content were not obvious. TDZ was important for callus induction. The bigger volume of callus and the higher induction rate were achieved on the medium containing TDZ and auxin (IBA, NAA and 2,4-D).(3) The results of different concentrations of gibberellin showed:The maximum diameter of callus was 0.5 cm and the colour was faint yellow when the MS medium supplied 0.0050 mmol/L gibberellin, and the maximum callus induction rate was 81.250% at this concentration. The highest total flavonoids content was 3.420%(0.0025 mmol/L). Spermine was not easily promote the callus formation. The highest callus induction rate (19.167%), and the maximum dry weight (0.243 g) were obtained in the medium with 0.5 mmol/L spermine. The highest total flavonoids content was 2.857%(1.0 mmol/L), higher than the other groups.(4) When different concentrations of sucrose were supplied in MS medium, the efficient callus induction (49.917%) and the maximum dry weight (0.150 g) were obtained on MS medium with 2% sucrose. And the highest total flavonoids content was 2.762%(3% sucrose).